Some errors in the colorimetric estimation of oestriol, oestrone and oestradiol by the Kober reaction

Abstract

In the course of an investigation of methods suitable for the accurate determination of oestriol, oestrone and oestradiol-17ft in the urine of human subjects, it was found that the colour produced in the Kober reaction by oestrogens was depressed by 15 40% in the presence of solvent residues. The particular modification of the Kober (1931) reaction which was principally used in this investigation was one using quinol and aqueous sulphuric acid (Brown, 1952), but the same effect was found with the original phenol reagent of Kober under the condi- tions described by Venning, Evelyn, Harkness & Browne (1937). The inhibition of colour production may readily be demonstrated by the addition to a pure oestrogen of the residue from distillation of ether which has been used to extract dilute mineral acid and washed with bicarbonate and water (as is required in the initial extraction of oestrogens from hydrolysed urine). The effect persisted even when the ether was purified by a variety of methods. The magnitude of error involved was sufficient to necessitate a thorough re-investigation of the colour reaction described by Brown (1952).